1. Technical Field
The present invention relates to methods and compositions for producing a transgenic mammal which expresses an exogenously supplied gene in lung tissue. The gene is supplied by aerosolized delivery, particularly to the airways and alveoli of the lung.
2. Background
With the advent of molecular cloning techniques, an expanding array of genes with mutations responsible for important human diseases have been identified and isolated. To date, attempts to replace absent or mutated genes in human patients have relied on ex vivo techniques. Ex vivo techniques include transformation of cells in vitro with either naked DNA or DNA encapsulated in liposomes, followed by introduction into a host organ (xe2x80x9cex vivoxe2x80x9d gene therapy). The criteria for a suitable organ include that the target organ for implantation is the site of the relevant disease, the disease is easily accessible, that it can be manipulated in vitro, that it is susceptible to genetic modification methods and ideally, it should contain either non-replicating cells or cycling stem cells to perpetuate a genetic correction. It also should be possible to reimplant the genetically modified cells into the organism in a functional and stable form. A farther requirement for ex vivo gene therapy, if for example a retroviral vector is used, is that the cells be pre-mitotic; post-mitotic cells are refractory to infection with retroviral vectors. Exemplary of a target organ which meets the criteria of in vitro gene transfer is the mammalian bone marrow.
There are several drawbacks to ex vivo therapy. For example, if only differentiated, replicating cells are infected, the newly introduced gene function will be lost as those cells mature and die. Ex vivo approaches also can be used to transfect only a limited number of cells and cannot be used to transfect cells which are not first removed from the body.
Retroviruses, adenoviruses and liposomes have been used in animal model studies in attempts to increase the efficiency of gene transfer; DNA has been introduced into animals by intratracheal (IT), intravenous, intraperitoneal, intramuscular, and intraarterial injection. Expression of introduced genes, either complexed to cationic liposomes or packaged in adenoviral vectors has been demonstrated in the lungs of rodents after IT instillation. However, IT injection is invasive and produces a non-uniform distribution of the instilled material; it also is too invasive to be performed repeatedly in humans. It therefore would be of interest to develop a non-invasive delivery technique which also results in deeper penetration of material into the lung than other methods, and can be used to deposit material evenly throughout the airways and alveoli. Such a delivery technique could be used as a means of treatment for genetic disorders, particularly of the lung, via generalized transgene expression in lung cells in vivo.
Relevant Literature
Hazinski, et al., Am. J. Respir. Cell Mol. Biol. (1991) 4:206-209, relates to liposome-mediated gene transfer of DNA into the intact rodent lung. Three fusion gene constructs were complexed to cationic liposomes including (1) the chloramphenicol acetyltransferase (xe2x80x9cCATxe2x80x9d) gene linked to a Rous sarcoma virus (xe2x80x9cRSVxe2x80x9d) promoter; (2) the CAT gene linked to a mouse mammary tumor virus (xe2x80x9cMMTVxe2x80x9d) promoter; and (3) a cytomegalovirus-xcex2-galactosidase (xe2x80x9cCMV-xcex2-galxe2x80x9d) fusion gene. The liposome/DNA complexes were instilled into the cervical trachea of rats and detectable levels of gene expression observed. Brigham et al, Am. J. Med. Sci. (1989) 298:278-281, describes the in vivo transfection of murine lungs with the CAT gene using a liposome vehicle. Transfection was accomplished by intravenous, intratracheal or intraperitoneal injection. Both intravenous and intratracheal administration resulted in the expression of the CAT gene in the lungs. However, intraperitoneal administration did not. Canonico et al., Clin. Res. (1991) 39:219A describes the expression of the human xcex1-1 antitrypsin gene, driven by the CMV promoter, in cultured bovine lung epithelial cells. The gene was added to cells in culture using cationic liposomes. The experimenters also detected the presence of xcex1-1 antitrypsin in histological sections of the lung of New Zealand white rabbits following the intravenous delivery of gene constructs complexed to liposomes. Wolff et al., Science (1990) 247:1465-1468 relates to direct transfer of the CAT, xcex2-gal and luciferase genes into mouse skeletal muscle in vivo. Gene expression was observed in all three cases. Nabel et al., Science (1990) 249:1285-1288, pertains to in vivo intra-arterial transfection of pigs with liposomes containing a xcex2-gal expression plasmid. Site-specific gene expression was observed in the arterial wall. None of the above cited art, however, practices or teaches the use of aerosol administration to deliver genes directly to the lung.
PCT/US90/01515, having International Publication No. WO 90/11092, describes a method for introducing naked DNA into muscle tissue. Yoshimura et al. disclose expression of the human cystic fibrosis transmembrane conductance regulator gene in mouse lung after intratracheal plasmid-mediated gene transfer. Debs et al. disclose pentamidine uptake in the lung by aerosolization and delivery in liposomes. Am Rev Respir Dis (1987) 135: 731-737.
Methods and compositions are provided for producing a mammal which expresses an exogenously supplied gene of interest in cells of the lung. The method includes the steps of preparing a liposome-nucleic acid mixture suitable for nebulization, nebulizing the mixture, and depositing the resulting nebulized mixture in the lung of a mammalian host of interest in an amount sufficient to transform cells contacted by the deposited nebulized mixture. The exogenously supplied gene generally is provided in an expression cassette and includes a coding sequence operably joined to transcriptional and translational regulatory sequences functional in the mammalian host. The methods and compositions find use as in vivo gene therapy of pulmonary disorders.